Flow cytometry is a high-throughput measurement method for examining fluorescence in cells or particles or other items. However, as recognized by the present inventors, typically flow cytometers only screen cells on the basis of fluorescence intensity and cannot be used to examine the photobleaching of fluorescent proteins in cells, and conventional flow cytometers cannot typically be used to perform time-resolved and repeated measurements of fluorescent particles or cells.
Moreover, fluorescence photobleaching is a complex phenomenon that depends on several experimental and environmental factors. In fluorescent proteins, photobleaching occurs by both reversible and irreversible pathways, both of which may limit photon output of the fluorescent proteins. For instance, under single-molecule excitation conditions, fluorescent proteins emit 10-100× fewer photons than small-molecule fluorophores.
Fluorescent proteins (FPs) have revolutionized cellular imaging, however, they have a tendency to convert to non-radiative dark states and to undergo rapid, irreversible photobleaching. These effects limit their use in imaging applications involving low concentrations or long observation times.